The goal of this grant continues to be the elucidation of the mechanisms of inflammation in allergic diseases of the airways and the mechanisms by which glucocorticoids ameliorate allergic disease. Based upon recent findings in the laboratory, we have advanced three important hypotheses that will be tested by work described in this proposal. The first hypothesis is that direct inhibition of STAT6 in epithelial cells will produce anti-inflammatory effects that exceed or supplement those of glucocorticoids. Epithelial expression of STAT6-dependent genes is a centrally important event in allergic inflammation, and recent studies indicate that glucocorticoids have no direct effect on STAT6 signaling in epithelial cells. Experiments are proposed which will develop new methodologies to inhibit STAT6 in airway epithelial cells to test this hypothesis. The second hypothesis is that airway epithelial cells manifest a local equivalent of the acute phase response following stimulation with ligands for Toll-like receptors (TLR) and inflammatory cytokines. Increased expression of complement proteins, pentraxins, collectin molecules and other acute phase reactants by epithelium may participate in an active host defense response. Integrated microarray, gene ontology-based, analytical and functional studies will characterize the innate host defense molecules expressed by activated epithelial cells. Whether epithelial cells exert this response in vivo will be tested in normal human subjects and those with inflammatory diseases of the upper airways. The third hypothesis is that glucocorticoids inhibit inflammatory responses but spare, or even enhance, innate immune responses. Preliminary results and published literature support this hypothesis, which will be tested using a systematic analysis of glucocorticoid regulation of gene expression. Parallel studies will test the hypothesis that epithelial activation by TLR ligands, cytokines and glucocorticoids involves the CCAAT-enhancer binding protein (C/EBP) family of transcription factors. Whether glucocorticoids enhance the expression of innate host defense molecules will be tested in vivo in human subjects using well-established techniques to collect and analyze airway epithelial responses. The studies described in this proposal are likely to be of great relevance to the clinical use of glucocorticoids and may identify new strategies to develop drugs that will supplant or supplement glucocorticoids in the management of allergic disease of the airways.